Integrative visual omics of the white-rot fungus Polyporus brumalis exposes the biotechnological potential of its oxidative enzymes for delignifying raw plant biomass

AbstractWhite-rot fungi are wood decayers able to degrade all polymers from lignocellulosic biomass including cellulose, hemicelluloses, and lignin. The white-rot fungus Polyporus brumalis efficiently breaks down lignin and is regarded as having a high potential for the initial treatment of plant biomass in its conversion to bio-energy. We performed integrative multi-omics analyses by combining data from the fungal genome, transcriptomes, and secretomes. We found the fungus possessed an unexpectedly large set of genes coding for enzymes related to lignin degradation, and that these were highly expressed and massively secreted under solid-state fermentation conditions. The examination of interrelated multi-omics patterns revealed the coordinated regulation of lignin-active peroxidases and H2O2-generating enzymes along with the activation of cellular mechanisms for detoxification, which combined to result in the efficient lignin breakdown by the fungus.ImportancePlant biomass conversion for green chemistry and bio-energy is a current challenge for a modern sustainable bioeconomy. The complex polyaromatic lignin polymers in raw biomass feedstocks (i.e. agriculture and forestry by-products) are major obstacles for biomass conversions. From a biotechnological aspect, these compounds could be a potential source of aromatic platform molecules for bio-based polymers. Here we describe the extraordinary ability of Polyporus brumalis for lignin degradation using its enzymatic arsenal to break down wheat straw, a lignocellulosic substrate that is considered as a biomass feedstock worldwide. We observed unusual expansions of gene families coding for; 1) Class II peroxidases involved in lignin degradation; and 2) GMC oxidoreductases/dehydrogenases involved in generating the hydrogen peroxide required for lignin peroxidase activity. Our findings suggested the fungus massively mobilizes this oxidative machinery during growth on wheat straw. Overall, we identified sets of co-regulated enzymes, which could potentially augment the efficiency of biotechnological plant biomass conversions.

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Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus Dichomitus squalens

Biomolecules ◽

10.3390/biom11101526 ◽

2021 ◽

Vol 11 (10) ◽

pp. 1526

Author(s):

Joanna E. Kowalczyk ◽

Shreya Saha ◽

Miia R. Mäkelä

Keyword(s):

Genome Editing ◽

Plant Biomass ◽

Genetic Manipulation ◽

White Rot ◽

White Rot Fungus ◽

Detailed Knowledge ◽

Wild Type ◽

Low Frequencies ◽

Dichomitus Squalens

Dichomitus squalens is an emerging reference species that can be used to investigate white-rot fungal plant biomass degradation, as it has flexible physiology to utilize different types of biomass as sources of carbon and energy. Recent comparative (post-) genomic studies on D. squalens resulted in an increasingly detailed knowledge of the genes and enzymes involved in the lignocellulose breakdown in this fungus and showed a complex transcriptional response in the presence of lignocellulose-derived compounds. To fully utilize this increasing amount of data, efficient and reliable genetic manipulation tools are needed, e.g., to characterize the function of certain proteins in vivo and facilitate the construction of strains with enhanced lignocellulolytic capabilities. However, precise genome alterations are often very difficult in wild-type basidiomycetes partially due to extremely low frequencies of homology directed recombination (HDR) and limited availability of selectable markers. To overcome these obstacles, we assessed various Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) -based strategies for selectable homology and non-homologous end joining (NHEJ) -based gene editing in D. squalens. We also showed an induction of HDR-based genetic modifications by using single-stranded oligodeoxynucleotides (ssODNs) in a basidiomycete fungus for the first time. This paper provides directions for the application of targeted CRISPR/Cas9-based genome editing in D. squalens and other wild-type (basidiomycete) fungi.

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Irpex lacteus, a white-rot fungus with biotechnological potential — review

Folia Microbiologica ◽

10.1007/s12223-009-0053-2 ◽

2009 ◽

Vol 54 (5) ◽

pp. 375-390 ◽

Cited By ~ 63

Author(s):

Č. Novotný ◽

T. Cajthaml ◽

K. Svobodová ◽

M. Šušla ◽

V. Šašek

Keyword(s):

White Rot ◽

White Rot Fungus ◽

Irpex Lacteus ◽

Biotechnological Potential

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Influence of white-rot fungus Polyporus brumalis BRFM 985 culture conditions on the pretreatment efficiency for anaerobic digestion of wheat straw

Biomass and Bioenergy ◽

10.1016/j.biombioe.2018.01.018 ◽

2018 ◽

Vol 110 ◽

pp. 75-79 ◽

Cited By ~ 14

Author(s):

Elsa Rouches ◽

Simeng Zhou ◽

Michelle Sergent ◽

Sana Raouche ◽

Helene Carrere

Keyword(s):

Anaerobic Digestion ◽

Wheat Straw ◽

Culture Conditions ◽

White Rot ◽

White Rot Fungus ◽

Polyporus Brumalis

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Dibutyl phthalate biodegradation by the white rot fungus,Polyporus brumalis

Biotechnology and Bioengineering ◽

10.1002/bit.21333 ◽

2007 ◽

Vol 97 (6) ◽

pp. 1516-1522 ◽

Cited By ~ 30

Author(s):

Soo-Min Lee ◽

Jae-Won Lee ◽

Bon-Wook Koo ◽

Myung-Kil Kim ◽

Don-Ha Choi ◽

...

Keyword(s):

Dibutyl Phthalate ◽

White Rot ◽

White Rot Fungus ◽

Polyporus Brumalis

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Induction of Genes Encoding Plant Cell Wall-Degrading Carbohydrate-Active Enzymes by Lignocellulose-Derived Monosaccharides and Cellobiose in the White-Rot FungusDichomitus squalens

Applied and Environmental Microbiology ◽

10.1128/aem.00403-18 ◽

2018 ◽

Vol 84 (11) ◽

Cited By ~ 11

Author(s):

Sara Casado López ◽

Mao Peng ◽

Tedros Yonatan Issak ◽

Paul Daly ◽

Ronald P. de Vries ◽

...

Keyword(s):

Cell Wall ◽

Plant Cell ◽

Plant Cell Wall ◽

Plant Biomass ◽

Expression Patterns ◽

Fine Tuning ◽

White Rot ◽

White Rot Fungus ◽

Content Type ◽

Dichomitus Squalens

ABSTRACTFungi can decompose plant biomass into small oligo- and monosaccharides to be used as carbon sources. Some of these small molecules may induce metabolic pathways and the production of extracellular enzymes targeted for degradation of plant cell wall polymers. Despite extensive studies in ascomycete fungi, little is known about the nature of inducers for the lignocellulolytic systems of basidiomycetes. In this study, we analyzed six sugars known to induce the expression of lignocellulolytic genes in ascomycetes for their role as inducers in the basidiomycete white-rot fungusDichomitus squalensusing a transcriptomic approach. This identified cellobiose andl-rhamnose as the main inducers of cellulolytic and pectinolytic genes, respectively, ofD. squalens. Our results also identified differences in gene expression patterns between dikaryotic and monokaryotic strains ofD. squalenscultivated on plant biomass-derived monosaccharides and the disaccharide cellobiose. This suggests that despite conservation of the induction between these two genetic forms ofD. squalens, the fine-tuning in the gene regulation of lignocellulose conversion is differently organized in these strains.IMPORTANCEWood-decomposing basidiomycete fungi have a major role in the global carbon cycle and are promising candidates for lignocellulosic biorefinery applications. However, information on which components trigger enzyme production is currently lacking, which is crucial for the efficient use of these fungi in biotechnology. In this study, transcriptomes of the white-rot fungusDichomitus squalensfrom plant biomass-derived monosaccharide and cellobiose cultures were studied to identify compounds that induce the expression of genes involved in plant biomass degradation.

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Glucose-Mediated Repression of Plant Biomass Utilization in the White-Rot Fungus Dichomitus squalens

Applied and Environmental Microbiology ◽

10.1128/aem.01828-19 ◽

2019 ◽

Vol 85 (23) ◽

Cited By ~ 2

Author(s):

Paul Daly ◽

Mao Peng ◽

Marcos Di Falco ◽

Anna Lipzen ◽

Mei Wang ◽

...

Keyword(s):

Catabolite Repression ◽

Carbon Catabolite Repression ◽

Plant Biomass ◽

Carbon Sources ◽

White Rot Fungi ◽

White Rot ◽

White Rot Fungus ◽

Content Type ◽

Dichomitus Squalens ◽

Genes Encoding

ABSTRACT The extent of carbon catabolite repression (CCR) at a global level is unknown in wood-rotting fungi, which are critical to the carbon cycle and are a source of biotechnological enzymes. CCR occurs in the presence of sufficient concentrations of easily metabolizable carbon sources (e.g., glucose) and involves downregulation of the expression of genes encoding enzymes involved in the breakdown of complex carbon sources. We investigated this phenomenon in the white-rot fungus Dichomitus squalens using transcriptomics and exoproteomics. In D. squalens cultures, approximately 7% of genes were repressed in the presence of glucose compared to Avicel or xylan alone. The glucose-repressed genes included the essential components for utilization of plant biomass—carbohydrate-active enzyme (CAZyme) and carbon catabolic genes. The majority of polysaccharide-degrading CAZyme genes were repressed and included activities toward all major carbohydrate polymers present in plant cell walls, while repression of ligninolytic genes also occurred. The transcriptome-level repression of the CAZyme genes observed on the Avicel cultures was strongly supported by exoproteomics. Protease-encoding genes were generally not glucose repressed, indicating their likely dominant role in scavenging for nitrogen rather than carbon. The extent of CCR is surprising, given that D. squalens rarely experiences high free sugar concentrations in its woody environment, and it indicates that biotechnological use of D. squalens for modification of plant biomass would benefit from derepressed or constitutively CAZyme-expressing strains. IMPORTANCE White-rot fungi are critical to the carbon cycle because they can mineralize all wood components using enzymes that also have biotechnological potential. The occurrence of carbon catabolite repression (CCR) in white-rot fungi is poorly understood. Previously, CCR in wood-rotting fungi has only been demonstrated for a small number of genes. We demonstrated widespread glucose-mediated CCR of plant biomass utilization in the white-rot fungus Dichomitus squalens. This indicates that the CCR mechanism has been largely retained even though wood-rotting fungi rarely experience commonly considered CCR conditions in their woody environment. The general lack of repression of genes encoding proteases along with the reduction in secreted CAZymes during CCR suggested that the retention of CCR may be connected with the need to conserve nitrogen use during growth on nitrogen-scarce wood. The widespread repression indicates that derepressed strains could be beneficial for enzyme production.

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